There is increasing evidence that the mosquito midgut mounts immune responses to the malaria parasite during invasion. However, the ookinete is able to survive these responses to eventually be transmitted to the vertebrate host. The general goal of this proposal is to gain new insight into the mechanisms involved in this process. We propose to study the activation of two immuno-regulatory pathways in the midgut and fat body. Electrophoretic mobility shift assays (EMSA) will be used to follow the induction of DNA binding activity to STAT-like and NF-kappaB-like target sequences. Our preliminary experiments indicate that the midgut is the main organ activating the JAK-STAT pathway in response to malaria infection. The time course of these responses will be also analyzed. We will study the midgut epithelium immune responses to ookinete invasion at the cellular level by analyzing the pattern of expression, sub-cellular localization and the induction of different immune markers by immunocytochemistry. We will determine if genes known to be induced by malaria infection are regulated by the JAK-STAT pathway. A cDNA subtraction strategy is proposed to isolate new malaria inducible genes. Recent Southern blot analysis of Ag-STAT BAC clones suggests that a new STAT family member is located in close physical proximity to Ag-STAT. We will attempt to characterize and study this new gene. Establishing which cells are invaded by the ookinete and the molecular nature of their responses is central to understanding the survival strategy of the parasite.